TW202023613A - Anti-claudin182 antibody and use thereof - Google Patents

Abstract

Provided are an anti-Claudin18.2 antibody or an antigen binding fragment thereof, a pharmaceutical composition comprising an anti-Claudin18.2 antibody or an antigen binding fragment thereof, and uses thereof. The antibody and Claudin18.2 have significant binding activity and affinity, have efficient in-vitro ADCC cytotoxic activity against a tumor cell, and can be applied in the preparation of an antineoplastic drug.

Description

An anti-Claudin18_2 antibody and its application

The present invention relates to the technical field of antibody medicines, in particular to an anti-Claudin 18.2 antibody or an antigen-binding fragment thereof, a pharmaceutical composition comprising an anti-Claudin 18.2 antibody or an antigen-binding fragment thereof, and their applications.

Gastric cancer is the fifth most common type of cancer in the world, and its fatality rate is the third highest. Currently, chemotherapy is the standard first-line treatment for advanced or recurrent gastric cancer. More than 90% of cancer patients will receive ordinary chemotherapy and targeted chemotherapy, but chemotherapy generally cannot cure cancer. It can only prolong patient survival and improve the quality of life, and it is prone to drug resistance. Biological targeted therapy usually targets tumor-specific antigens or tumor-related antigens, and relies on the binding or blocking of antibodies to the target to trigger the body's immune response and selectively kill the tumor without affecting the surrounding normal cells and tissues. Targeted therapy has strong specificity, few side effects and definite curative effect.

However, due to the heterogeneity of malignant tumors, different cancers show inconsistent molecular biological characteristics. Especially for gastric cancer, the degree of heterogeneity is relatively high. The bio-targeted therapeutic drugs developed for gastric cancer in the past are basically ineffective as a single drug or effective for a very small number of patients. The reason is that the targets targeted by these monoclonal antibodies are only expressed in specific subtypes of gastric cancer patients. A typical example is Trastuzumab, which has a good effect in combination with chemotherapy, but it can only be applied to HER2-positive individuals who account for only 15% of all gastric cancer patients.

Claudins are the most important skeletal proteins that determine the structure of tight junctions between cells. They participate in adhesion junctions and play an important role in tumor cell metastasis and invasion. Claudin protein is widely present in mammalian epithelial and endothelial cells, and its distribution is mainly on the side of epithelial cells and the plasma membrane of basal cells.

Different Claudin proteins have their own specific expressions in different tissues. The Claudin18 (CLDN18) gene is located at 3q22.3, has a molecular weight of 24kDa, and contains 261 amino acid residues. It is a member of the Claudins superfamily. Its protein structure contains 2 extracellular Ring and 4 penetrating zones. The two subtypes of human CLDN18 protein are CLDN18.1 (NM_016369.3) and CLDN18.2 (NM_001002026.2). In the primary structure sequence of the two proteins, only the N-terminal signal peptide to the extracellular loop 1 (Loop1 ) There are differences in amino acid residues in certain positions of the structure, especially in the extracellular loop 1. CLDN18.1 and CLDN18.2 differ only by 8 amino acids. In addition, the sequence homology between the two subtype proteins of CLDN18 is also very high. Among them, the extracellular loop 1 of CLDN18.2 has the same sequence in different species such as humans, mice, and rhesus monkeys, while the CLDN18.2 protein homology between human and mouse reaches 84%, indicating that the CLDN18.2 protein sequence is extremely conservative (O.Tureci., et al., Gene 481: 83-92, 2011).

The two subtype proteins of CLDN18 are expressed in different tissues and exert their physiological functions. CLDN18.1 is constitutively expressed in normal lung tissue cells; CLDN18.2 protein is expressed in normal gastric epithelial cell membranes, and is not expressed or very low in various tissues except the stomach. Salin et al. (U. Salin., et al., Clin Cancer Res 14(23): 7624-34, 2008) used RT-PCR and immunohistochemical methods to detect CLDN18 for a variety of cancer tissues including gastric cancer, pancreatic cancer, and esophageal cancer. .2 Positive, the overall positive rate reached 62.7%. Other studies have shown that CLDN18.2 protein expressed by normal parietal cells has uniform glycosylation modification and a compact structure between cells. The structure of gastric cancer cells is loose, and the cells are easy to invade and metastasize. The CLDN18.2 protein glycosylation on it is incomplete, which may lead to target exposure (WO2004/047863). Therefore, CLDN18.2 may be an ideal tumor-associated antigen target.

Currently, Astellas Pharma Inc. has developed a monoclonal antibody drug Zolbetuximab (also known as IMAB362) that targets human Claudin 18.2 and has significant clinical efficacy. Phase II clinical trials (S. Batran. et al., Journal of Clinical Oncology 34 (no.18_suppl), 2016) showed that the combination of monoclonal antibodies and chemical drugs in the treatment of advanced or recurrent gastric cancer has a median survival of 13.2 patients Months, significantly longer than 8.4 months with chemotherapy alone. It is preliminarily confirmed that specific antibodies targeting CLDN18.2 can significantly improve the progression-free survival and overall survival of patients with advanced gastric and gastroesophageal junction adenocarcinoma.

WO2004/047863 describes the preparation of a human-mouse chimeric monoclonal antibody that is the world's first therapeutic antibody developed against the human Claudin 18.2 target (hereinafter referred to as the anti-CLDN18.2 antibody). The antibody described in the patent application specifically kills CLDN18.2 positive tumor cells mainly through the antibody-dependent cell-mediated cytotoxicity (ADCC) pathway. However, the specific recognition and killing between the antibody described in the patent application and tumor cells mainly depends on the abundance of the CLDN18.2 target on the tumor cells, which is more obvious when the cell surface is high on CLDN18.2. The binding and killing activity. This shows that it is necessary to prepare a high-affinity monoclonal antibody to improve its ability to selectively kill tumor cells.

Based on the above prior art, the present invention now provides a highly effective CLDN18.2 antibody or antigen-binding fragment thereof, which has excellent anti-tumor activity.

One aspect of the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region and/or a light chain variable region, wherein the heavy chain variable region comprises the complementarity determination of the heavy chain variable region Region 1 (HCDR1), the complementarity determining region 2 (HCDR2) of the heavy chain variable region and/or the complementarity determining region 3 (HCDR3) of the heavy chain variable region, the light chain variable region includes the light chain variable region Complementarity determining region 1 (LCDR1), complementarity determining region 2 (LCDR2) of the light chain variable region and/or complementarity determining region 3 (LCDR3) of the light chain variable region.

In some embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein: (1) The heavy chain variable region includes HCDR1, HCDR2 and HCDR3: (A1) The amino acid sequence of the HCDR1 is selected from SEQ ID NO 101, SEQ ID NO 102, SEQ ID NO 103 and an amino acid sequence having at least 85% sequence identity with them; (A2) The amino acid sequence of the HCDR2 is selected from SEQ ID NO 104, SEQ ID NO 105 and amino acid sequences having at least 85% sequence identity with them; (A3) The amino acid sequence of the HCDR3 is selected from SEQ ID NO 106, SEQ ID NO 107 and an amino acid sequence having at least 85% sequence identity with them; and (2) The light chain variable region includes LCDR1, LCDR2 and LCDR3, wherein: (A4) The amino acid sequence of LCDR1 is SEQ ID NO 108; (A5) The amino acid sequence of LCDR2 is selected from the group consisting of SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 111 and amino acid sequences having at least 85% sequence identity with them; (A6) The amino acid sequence of LCDR3 is selected from the group consisting of SEQ ID NO 112, SEQ ID NO 113, SEQ ID NO 114, SEQ ID NO 115, SEQ ID NO 116 and amino acid sequences having at least 85% sequence identity with them.

In a specific embodiment, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, which has the HCDR1, HCDR2, and HCDR3 as SEQ ID NO 101, 105 and 106, respectively, or with SEQ ID NO 101, 105 The amino acid sequence shown in and 106 has a heavy chain variable region of a CDR with at least 85% sequence identity, and the LCDR1, LCDR2, and LCDR3 are respectively SEQ ID NOs 108, 111 and 115 or with SEQ ID NOs 108, 111 and The amino acid sequence shown in 115 has a CDR light chain variable region with at least 85% sequence identity.

In some specific embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof according to the present invention is a monoclonal antibody or antigen-binding fragment thereof.

In some specific embodiments, the anti-CLDN18.2 antibody or antigen-binding fragment thereof according to the present invention is a murine antibody or an antigen-binding fragment thereof, a chimeric antibody or an antigen-binding fragment thereof, or a humanized antibody or an antigen-binding fragment thereof .

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein (1) The amino acid sequence of the variable region of the heavy chain is selected from: (B1) The amino acid sequence as shown in SEQ ID NO 50, SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO 77, SEQ ID NO 79; (B2) The amino acid sequence shown in (b1) is obtained by substituting, deleting or adding one or more amino acids and having the same or similar function as the amino acid sequence shown in (b1); and (B3) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (b1); and (2) The amino acid sequence of the light chain variable region is selected from: (B4) As shown in SEQ ID NO 51, SEQ ID NO 70, SEQ ID NO 71, SEQ ID NO 73, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 78, SEQ ID NO 80, SEQ ID NO 81 Amino acid sequence of (B5) The amino acid sequence shown in (b4) is obtained by substituting, deleting or adding one or more amino acids, and having the same or similar function as the amino acid sequence shown in (b4); and (B6) An amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (b4).

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 50, and SEQ ID NO: 50 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 50 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 50, and the amino acid sequence of the light chain variable region It is SEQ ID NO: 51, SEQ ID NO: 51 is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 51 or has at least 85% sequence identity with SEQ ID NO: 51 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 50, and SEQ ID NO: 50 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 50 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 50, and the amino acid sequence of the light chain variable region It is SEQ ID NO: 70, SEQ ID NO: 70 is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 70 or has at least 85% sequence identity with SEQ ID NO: 70 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 50, and SEQ ID NO: 50 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 50 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 50, and the amino acid sequence of the light chain variable region It is SEQ ID NO: 71, SEQ ID NO: 71 is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 71 or has at least 85% sequence identity with SEQ ID NO: 71 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 72, and SEQ ID NO: 72 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 72 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 72, and the amino acid sequence of the light chain variable region It is SEQ ID NO: 73, SEQ ID NO: 73 is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 73 or has at least 85% sequence identity to SEQ ID NO: 73 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 74, and SEQ ID NO: 74 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 74 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 74, and the amino acid sequence of the light chain variable region It is SEQ ID NO: 75, SEQ ID NO: 75 is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 75 or has at least 85% sequence identity to SEQ ID NO: 75 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 50, and SEQ ID NO: 50 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 50 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 50, and the amino acid sequence of the light chain variable region It is SEQ ID NO: 76, SEQ ID NO: 76 is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 76 or has at least 85% sequence identity with SEQ ID NO: 76 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 77, and SEQ ID NO: 77 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 77 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 77, and the amino acid sequence of the light chain variable region It is SEQ ID NO: 78, SEQ ID NO: 78 is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 78 or has at least 85% sequence identity with SEQ ID NO: 78 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 74, and SEQ ID NO: 74 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 74 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 74, and the amino acid sequence of the light chain variable region It is SEQ ID NO: 70, SEQ ID NO: 70 is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 70 or has at least 85% sequence identity with SEQ ID NO: 70 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 74, and SEQ ID NO: 74 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 74 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 74, and the amino acid sequence of the light chain variable region It is SEQ ID NO: 71, SEQ ID NO: 71 is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 71 or has at least 85% sequence identity with SEQ ID NO: 71 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 74, and SEQ ID NO: 74 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 74 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 74, and the amino acid sequence of the light chain variable region It is SEQ ID NO: 76, SEQ ID NO: 76 is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 76 or has at least 85% sequence identity with SEQ ID NO: 76 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 72, and SEQ ID NO: 72 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 72 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 72, and the amino acid sequence of the light chain variable region It is SEQ ID NO: 76, SEQ ID NO: 76 is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 76 or has at least 85% sequence identity with SEQ ID NO: 76 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 77, and SEQ ID NO: 77 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 77 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 77, and the amino acid sequence of the light chain variable region It is SEQ ID NO: 70, SEQ ID NO: 70 is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 70 or has at least 85% sequence identity with SEQ ID NO: 70 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 77, and SEQ ID NO: 77 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 77 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 77, and the amino acid sequence of the light chain variable region It is SEQ ID NO: 76, SEQ ID NO: 76 is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 76 or has at least 85% sequence identity with SEQ ID NO: 76 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 79, and SEQ ID NO: 79 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 79 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 79, and the amino acid sequence of the light chain variable region It is SEQ ID NO: 70, SEQ ID NO: 70 is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 70 or has at least 85% sequence identity with SEQ ID NO: 70 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 79, and SEQ ID NO: 79 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 79 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 79, and the amino acid sequence of the light chain variable region It is SEQ ID NO: 76, SEQ ID NO: 76 is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 76 or has at least 85% sequence identity with SEQ ID NO: 76 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 50, and SEQ ID NO: 50 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 50 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 50, and the amino acid sequence of the light chain variable region It is the amino acid sequence of SEQ ID NO: 80, SEQ ID NO: 80 obtained by substitution, deletion or addition of one or more amino acids and functionally identical to SEQ ID NO: 80 or at least 85% sequence identity to SEQ ID NO: 80 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 50, and SEQ ID NO: 50 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 50 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 50, and the amino acid sequence of the light chain variable region It is the amino acid sequence of SEQ ID NO: 81, SEQ ID NO: 81 is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 81 or has at least 85% sequence identity to SEQ ID NO: 81 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 79, and SEQ ID NO: 79 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 79 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 79, and the amino acid sequence of the light chain variable region It is the amino acid sequence of SEQ ID NO: 80, SEQ ID NO: 80 obtained by substitution, deletion or addition of one or more amino acids and functionally identical to SEQ ID NO: 80 or at least 85% sequence identity to SEQ ID NO: 80 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 79, and SEQ ID NO: 79 is substituted or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 79 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 79, and the amino acid sequence of the light chain variable region It is the amino acid sequence of SEQ ID NO: 81, SEQ ID NO: 81 is obtained by substitution, deletion or addition of one or more amino acids and is functionally identical to SEQ ID NO: 81 or has at least 85% sequence identity to SEQ ID NO: 81 Sexual amino acid sequence.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the antibody further contains the heavy chain constant region of human IgG1 or a variant thereof, a human kappa chain or The light chain constant region of the variant.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, in which the following amino acid substitutions are made in the variable region of the heavy chain whose amino acid sequence is as shown in SEQ ID NO 79: (C1) K3Q, L19R, R42G, P45L, A49S, E73D, T78S, E82Q, S84N, S88A, M93V, A118S, the amino acid sequence after replacement is shown in SEQ ID NO 82; (C2) K3Q, V5L, L19R, R42G, P45L, A49S, E73D, A75S, E82Q, S8N, S88A, M93V, A118S, the amino acid sequence after replacement is shown in SEQ ID NO 88.

And the amino acid sequence of the light chain variable region shown in SEQ ID NO 81 is replaced with the following amino acids: (C3) D1E, S9P, S10T, T12S, V13L, T14S, A15P, K18R, K21L, P49A, K51L, G63S, V64I, D66A, T69S, V84L, A86P, A106Q, L112I, the amino acid sequence after replacement is as SEQ ID NO 84 shown; (C4) S9D, T12A, T14S, A15L, K18R, V19A, M21I, S22N, T69S, V84L, L89V, A106Q, L112I, the amino acid sequence after replacement is shown in SEQ ID NO 86; (C5) A9L, T12P, A15L, E17Q, K18P, V19A, T20S, M21I, K45R, P49S, K51R, T69S, T80K, S83R, Q85E, L89V, A90G, A106Q, L112I, the amino acid sequence after replacement is as SEQ ID NO 90 shown.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein: (1) The amino acid sequence of the variable region of the heavy chain is selected from: (C1) The amino acid sequence shown in SEQ ID NO 82 and SEQ ID NO 88; (C2) The amino acid sequence shown in (c1) is obtained by substituting, deleting or adding one or more amino acids, and having the same or similar function as the amino acid sequence shown in (c1); and (C3) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (c1); and (2) The amino acid sequence of the light chain variable region is selected from: (C4) The amino acid sequences shown in SEQ ID NO 84, SEQ ID NO 86, and SEQ ID NO 90; (C5) The amino acid sequence shown in (c4) is obtained by substituting, deleting or adding one or more amino acids and having the same or similar function as the amino acid sequence shown in (c4); and (C6) An amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (c4).

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain variable region is SEQ ID NO 82, and SEQ ID NO 82 is substituted, deleted or added. An amino acid sequence obtained from or multiple amino acids and having the same function as SEQ ID NO 82 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 82, and the amino acid sequence of the light chain variable region is SEQ ID NO 84 , SEQ ID NO 84 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO 84 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 84.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain variable region is SEQ ID NO 82, and SEQ ID NO 82 is substituted, deleted or added. An amino acid sequence obtained from or multiple amino acids and having the same function as SEQ ID NO 82 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 82, and the amino acid sequence of the light chain variable region is SEQ ID NO 86 , SEQ ID NO 86 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO 86 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 86.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, the amino acid sequence of the heavy chain variable region is SEQ ID NO 88, and SEQ ID NO 88 is substituted, deleted or added. An amino acid sequence obtained from or multiple amino acids and having the same function as SEQ ID NO 88 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 88, and the amino acid sequence of the light chain variable region is SEQ ID NO 84 , SEQ ID NO 84 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO 84 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 84.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, the amino acid sequence of the heavy chain variable region is SEQ ID NO 88, and SEQ ID NO 88 is substituted, deleted or added. The amino acid sequence obtained from multiple amino acids and which has the same function as SEQ ID NO 88 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 88, and the amino acid sequence of the light chain variable region is SEQ ID NO 86 , SEQ ID NO 86 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO 86 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 86.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, the amino acid sequence of the heavy chain variable region is SEQ ID NO 82, and SEQ ID NO 82 is substituted, deleted or added. An amino acid sequence obtained from or multiple amino acids and having the same function as SEQ ID NO 82 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 82, and the amino acid sequence of the light chain variable region is SEQ ID NO 90 An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO 90 and having the same function as SEQ ID NO 90 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 90.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, the amino acid sequence of the variable region of the heavy chain is SEQ ID NO 88. SEQ ID NO 88 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO 88 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 88, and the amino acid sequence of the light chain variable region is SEQ ID NO 90, The amino acid sequence of SEQ ID NO 90 obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO 90 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 90.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, which comprises a heavy chain amino acid sequence of SEQ ID NO 92, and SEQ ID NO 92 is substituted, deleted or added with one or more amino acids The obtained amino acid sequence having the same function as SEQ ID NO 92 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 92, and the light chain amino acid sequence is SEQ ID NO 94, SEQ ID NO 94 is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO 94 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 94.

In some specific embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, which comprises a heavy chain amino acid sequence of SEQ ID NO 96, and SEQ ID NO 96 is substituted, deleted or added with one or more amino acids The obtained amino acid sequence having the same function as SEQ ID NO 96 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 96, and the light chain amino acid sequence is SEQ ID NO 98, and SEQ ID NO 98 is substituted, deleted or An amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO 98 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 98.

Another aspect of the present invention provides an isolated nucleotide, which encodes the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present invention.

In some specific embodiments, the present invention provides a nucleotide sequence encoding the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present invention, wherein: (1) The nucleotide sequence encoding the amino acid sequence of the heavy chain is shown in SEQ ID NO 93 or SEQ ID NO 97; (2) The nucleotide sequence encoding the amino acid sequence of the light chain is shown in SEQ ID NO 95 or SEQ ID NO 99.

In some specific embodiments, the present invention provides a nucleotide sequence encoding the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present invention, wherein: (1) The nucleotide sequence encoding the heavy chain amino acid sequence SEQ ID NO 92 is shown in SEQ ID NO 93; and (2) The nucleotide sequence encoding the light chain amino acid sequence SEQ ID NO 94 is shown in SEQ ID NO 95.

In some specific embodiments, the present invention provides a nucleotide sequence encoding the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present invention, wherein: (1) The nucleotide sequence encoding the heavy chain amino acid sequence SEQ ID NO 96 is shown in SEQ ID NO 97; and (2) The nucleotide sequence encoding the light chain amino acid sequence SEQ ID NO 98 is shown in SEQ ID NO 99.

Another aspect of the present invention provides a performance vector that expresses the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present invention. The expression vector according to the present invention contains the isolated nucleotide molecule of the present invention.

Another aspect of the present invention provides a host cell transformed with the expression vector as described above.

In some embodiments, the host cell according to the present invention is selected from prokaryotic cells and eukaryotic cells. In some embodiments, the host cell is bacteria, preferably Escherichia coli. In another preferred embodiment, the host cell is a mammalian cell.

Another aspect of the present invention provides a method for preparing the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present invention, including the steps of expressing the antibody in the host cell and isolating the antibody from the host cell.

Another aspect of the present invention provides a pharmaceutical composition comprising the anti-CLDN18.2 humanized antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the present invention provides a pharmaceutical composition comprising the anti-CLDN18.2 humanized antibody or antigen-binding fragment thereof of the present invention, and also other active components, such as other antibodies, targeted drugs, and the like. In some embodiments, the pharmaceutically acceptable carrier is selected from antioxidants, polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions, and surfactants. In a specific embodiment, the pharmaceutically acceptable carrier is a buffered aqueous solution. In another specific embodiment, the pharmaceutically acceptable carrier is in the form of liposomes.

The anti-CLDN18.2 humanized antibody or antigen-binding fragment thereof of the present invention can be mixed with a pharmaceutically acceptable carrier, diluent or excipient to prepare a pharmaceutical preparation suitable for oral or parenteral administration. Administration methods include, but are not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, intracerebral, intraocular, intratracheal, subcutaneous, and intranasal routes. The preparation can be administered by any route, for example, by infusion or bolus injection, by transepithelial or skin mucosa (for example, oral mucosa or rectum, etc.) absorption. Administration can be systemic or local. The formulation can be prepared by a method known in the art, and contains a carrier, diluent or excipient conventionally used in the field of pharmaceutical formulations.

Another aspect of the present invention provides a method of targeted CLDN18.2 therapy, the method comprising administering the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention to an individual in need thereof.

Another aspect of the present invention provides the use of the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention in the preparation of drugs targeting CLDN18.2. In some embodiments, the drug targeting CLDN 18.2 is used to treat gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer. In some embodiments, the present invention provides the use of the above-mentioned anti-CLDN18.2 antibody or antigen-binding fragment thereof or the pharmaceutical composition of the present invention in the preparation of anti-tumor drugs. Preferably, the tumor is selected from gastric cancer and esophageal cancer , Pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer and gallbladder cancer.

The anti-CLDN18.2 antibody or its antigen-binding fragment provided by the present invention has a significant anti-tumor effect, high affinity, strong ability of ADCC in vitro to kill tumor cells, and has significant technical advantages.

definition:

Unless otherwise defined, the meanings of scientific and technical terms used herein are those commonly understood by those skilled in the art. The nomenclature and techniques used in cell and tissue culture, molecular biology, and protein and oligo or polynucleotide chemistry and hybridization described herein are well known and commonly used in the art. For recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (eg electroporation, lipofection), standard techniques are used. The enzymatic reaction and purification techniques are performed according to the manufacturer's instructions or commonly used in the art or methods described herein. The aforementioned techniques and methods are generally used as described in multiple comprehensive and more specific documents that are well known in the art and cited and discussed in this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual) (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)). The nomenclature and laboratory methods and techniques used in analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are well known and commonly used in the art.

In the present invention, the term "at least 80% sequence identity" means at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity. In the present invention, the term "at least 85% sequence identity" means at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity. In some preferred embodiments, the sequence identity of the present invention may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. Sequence comparison and determination of percent identity between two sequences can be performed through the BLASTN/BLASTP algorithm on the National Center For Biotechnology Instutute website.

In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in a three-dimensional space to form an antigen binding surface. The antigen binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy chain and light chain are called "complementarity determining regions" or "CDRs". The assignment of amino acids to each domain is based on Kabat "Sequences of Proteins of Immunological Interest" (National Institutes of Health, Bethesda, Maryland (1987 and 1991)) or Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), Chothia et al., Nature 342:878-883 (1989) definition.

The "antigen-binding fragment" in the present invention refers to Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments and scFv fragments that bind to human Claudin 18.2 that have antigen-binding activity. The Fv fragment contains the variable region of the heavy chain of the antibody and the variable region of the light chain, but does not have the constant region, and has the smallest antibody fragment with all the antigen binding sites. Generally, Fv antibodies also include a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding. Different linkers can also be used to connect the variable regions of two antibodies into a single polypeptide chain, which is called single-chain antibody or single-chain Fv (scFv).

The antibody described in the present invention refers to an immunoglobulin molecule or an immunologically active part thereof, that is, a molecule containing an antigen binding site that specifically binds to (immunely reacts with) an antigen. "Specific binding" means that the antibody reacts with one or more antigenic determinants of the antigen but does not react with other polypeptides or binds to other polypeptides with very low affinity (Kd>10-6). Antibodies include, but are not limited to, multiple replication, single replication, chimeric, dAb (domain antibody), single chain, Fab, Fab' and F(ab') 2 fragments, Fv, scFv and Fab expression libraries. Monoclonal antibody (mAb) is an antibody obtained from a single replicating cell line, and the cell line is not limited to eukaryotic, prokaryotic or phage replicating cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombinant techniques such as hybridoma technology, recombination technology, phage display technology, and synthesis technology such as CDR grafting or other existing technologies.

The "murine antibody" of the present invention is a monoclonal antibody to human CLDN 18.2 prepared according to the knowledge and skills in the field. During the preparation, the test subject was injected with CLDN18.2 antigen, and then hybridomas exhibiting antibodies with desired sequences or functional properties were isolated.

The "chimeric antibody" of the present invention is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, it is necessary to first establish a hybridoma secreting murine-derived specific monoclonal antibodies, then copy the variable region genes from the mouse hybridoma cells, and then copy the constant region genes of the human antibody as needed. The gene and the human constant region gene are connected to form a chimeric gene and then inserted into the expression vector, and finally the chimeric antibody molecule is displayed in the eukaryotic industrial system or the prokaryotic industrial system.

The "humanized antibody" of the present invention is also called a CDR grafted antibody, and is an antibody produced by grafting mouse CDR sequences into a human antibody variable region framework (FR). Such variable region framework sequences can be obtained from public DNA databases or public references, for example, from the ImmunoGeneTics (IMGT) website http://imgt.cines.fr or from the Journal of Immunoglobulin, 2001ISBN012441351.

The following representative embodiments are used to better illustrate the present invention, rather than to limit the protection scope of the present invention. The experimental methods without conditions indicated in the following examples are usually carried out in accordance with conventional conditions, such as the antibody technology experiment manual and molecular replication manual of Cold Spring Harbor, or according to the conditions recommended by the raw material or commodity manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

Example 1 Preparation of positive control antibody

1. Preparation of positive control antibody IMAB362-similar-hG1:

According to the 175D10 replication (IMAB362) antibody sequence provided in the patent application WO2007/059997 (ie SEQ ID NO 11 and SEQ ID NO 12 in the present disclosure), Nanjing KingScript Biotechnology Co., Ltd. was entrusted to perform codon optimization and synthesized in pcDNA3. 1(+) In the vector. The constructed plastids include pcDNA3.1-hG1 and pcDNA3.1-hK. The plastids were co-transfected into expiCHO cells at a ratio of 1:1 for instantaneous performance, at 110 rpm, 37°C, 8% CO _{2 and} incubated for 4 days, then transferred to a 32°C shaker and cultured for 8-10 days. Centrifuge at 4500rpm for 25 minutes, and the protein supernatant was purified by ProteinA affinity chromatography: 0.1M NaOH solution to clean the AKTA chromatography system and column for 30 minutes, rinse with ultrapure water; pH7.4 20mM sodium phosphate buffer balance 10 Column volume; set flow rate 2 ml/min, load the sample; equilibrate the column to wash 10 column volumes; pH3.4 50mM acetate buffer elution 3-5 column volume, collect the eluted protein; ultrafiltration concentration The antibody was replaced in 20 mM phosphate buffer pH 7.0 and stored.

SDS-PAGE gel electrophoresis results showed that the molecular weight of the positive antibody IMAB362-similar-hG1 was about 155KD, and the SDS-PAGE purity was greater than 90%.

2. Preparation of mouse positive control antibody IMAB362-similar-mG2a:

That is, the antibody sequence SEQ ID NO 13 and SEQ ID NO 14 in which the variable region sequence of the positive antibody is combined with the constant region of mouse IgG2a. The codons were optimized and synthesized in pcDNA3.1(+) vector. The constructed plastids include pcDNA3.1-mG2a and pcDNA3.1-mK. The plastids were co-transfected into expiCHO cells at a ratio of 1:1 to express positive antibody protein transiently. The harvested supernatant protein was purified by ProteinA affinity chromatography: the AKTA chromatography system and the column were washed with 0.1M NaOH solution for 30 minutes, and then rinsed with ultrapure water; 20mM sodium phosphate buffer pH7.4 was used to balance 10 column volumes; set The flow rate is 2 ml/min, and the sample is loaded; the equilibration solution cleans the chromatography column for 10 column volumes; the 50mM acetate buffer with pH 3.4 elutes 3-5 column volumes to collect the eluted protein; ultrafiltration concentrates to replace the antibody at pH 7 .0 stored in 20mM phosphate buffer.

SDS-PAGE gel electrophoresis results showed that the non-reducing electrophoresis of the positive antibody IMAB362-similar-mG2a showed that the protein molecular weight was about 155KD, and the SDS-PAGE purity was greater than 90%.

Example 2 CLDN18.2 antigen gene synthesis and expression vector construction

The sequence design of the fusion protein expressing human CLDN18.2 antigen is shown in Table 1 below: Table 1 CLDN18.2 sequence design Serial number Sequence name Sequence meaning Amino acid sequence Nucleotide sequence 1 18A2-loop1 Sequence encoding CLDN18.2 extracellular loop 1 SEQ ID NO 2 SEQ ID NO 1 2 18A2-TM-Loop1 Sequence coding for extracellular loop 1 containing CLDN18.2 penetrating region SEQ ID NO 4 SEQ ID NO 3 3 CpG Unmethylated cytosine guanine dinucleotide palindrome sequence SEQ ID NO 5 4 CLDN18.2-FL Encoding human CLDN18.2 full-length protein sequence SEQ ID NO 8 SEQ ID NO 6 SEQ ID NO 7 5 CLDN18.1-FL Encoding human CLDN18.1 full-length protein sequence SEQ ID NO 10 SEQ ID NO 9

The fusion protein sequence of the human CLDN18.2 antigen designed above is optimized for eukaryotic codons, and restriction sites such as Not I and tPA signal peptide sequences are added at the 5'end of the gene, and restriction enzymes such as Xho I are added at the 3'end of the gene. Site and Myc-His fusion protein tag. The aforementioned SEQ ID NO 1, 3, 5, 6 sequences were synthesized and constructed into pcDNA3.1(+) vector to obtain DNA plastids expressing human CLDN18.2 antigen. The plastids include: pcDNA3.1-loop1, pcDNA3.1-TML, pcDNA3.1-CpG-loop1 and pcDNA3.1-CLDN18.2-FL.

The above-mentioned SEQ ID NO 7 and 9 sequences were optimized and synthesized by Bao Biological Engineering (Dalian) Co., Ltd. (hereinafter referred to as Bao Biological Company), and constructed on the lentiviral vector pLVX-NS-ZsGreen (Bao Biological Company). The constructed lentiviral plastids pLVX-CLDN18.2-ZsGreen and pLVX-CLDN18.1-ZsGreen were used to further construct the HEK293-CLDN18.2 fusion green fluorescent protein stable expression cell line, and to construct the HEK293-CLDN18.1 fusion green fluorescent protein Stable performance cell line.

The amplified and cultured plastids were used for restriction digestion identification, and the identification results showed that the digested bands were consistent with the expected size, indicating that the construction was correct.

Example 3 Expression of CLDN18.2 antigen protein

1. Transient transfection of plastid DNA in vitro

The plastids amplified and cultured in Example 2 were instantaneously transfected into HEK293T cells (Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, hereinafter referred to as Shanghai Institute of Cell Biology) in vitro, and the protein level was identified. The specific experimental steps are as follows: one day before transfection, HEK293T cells were plated in a six-well plate at a seeding density of 3×10 ⁵ cells/cm ² and cultured (medium composition: 10% fetal bovine serum DMEM medium + 1% penicillin- Streptomycin) overnight, make the cells fully adhere to the wall on the day of transfection, and make the cell abundance reach 70%-90% (the size of each well in the six-well plate is 9cm ² , and the cells are about 2-2.5×10 ⁶ cells/ Pores); use PEI cationic transfection reagent (sigma) or liposomes (Lipo3000, Thermo) to mix with DNA plastids to prepare DNA plastid transfection complex, add cells to room temperature and let stand for 5-20 minutes, 37℃, 5 Incubate in% CO ₂ for 4-5 hours; add complete medium containing serum to the six-well plate and culture for 48-72 hours to make the exogenous gene transiently express in eukaryotic cells in vitro.

2. Western blot hybridization

Collect transiently transfected HEK293T cells, hybridize the target protein with rabbit anti-human Claudin18 antibody (Thermo, Cat: 700178) at room temperature for 2 hours; wash 3 times with PBST, alkaline phosphatase labeled goat anti-rabbit IgG (Thermo, Cat: 31346) Hybridization at room temperature, 1 hour; Western Blot western blotting to identify the expression of the target protein: Using the Lipo liposome transfection system, the plastids can successfully express the target protein CLDN18.2-loop1 and CLDN18.2-TM-loop1, The molecular weights are 11KD and 14KD, respectively. The pcDNA3.1-CLDN18.2-FL plastid successfully expressed the target protein CLDN18.2 with a molecular weight of 29KD.

Example 4 Preparation of mouse hybridoma

1. Animal immunity:

According to the QIAGEN endotoxin-free plastid mass extraction kit instructions to extract and prepare plastid DNA, including pcDNA3.1-loop1 (hereinafter referred to as Loop1), pcDNA3.1-TM-Loop1 (hereinafter referred to as TML), pcDNA3.1-CpG-loop1 (Hereinafter referred to as Loop1-C). The plastids were grouped into mice according to the following experiment in Table 2. SJL mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and BALB/c mice and C57 mice were purchased from Huafukang Biological Products, Xuanwu District, Nanjing center. Immunization refers to the method described by Zhiwei Chen et al. (Z.Chen., et al., J Clin Invest. 123(6):2629-2642(2013)), with appropriate modifications, as follows: DNA plastids penetrate living bodies The gene transfer instrument (Shanghai Tarissa Biotechnology Co., Ltd.) was injected into the muscles of the left and right hind limbs of the mouse at two points, and the same amount of DNA plastids were injected at each point, with an injection volume of 50 µl. Immediately after the injection, the electrode of the importer was used to input electrical pulse stimulation to the injection site of the mouse, the voltage was set to 36V, and the pulse was 6 times in total. Electrical pulse stimulation can promote the uptake of DNA plastids by mouse muscle cells and increase the degree of mouse DNA immune response, especially humoral immune response. DNA immunization was injected every 2 weeks for a total of three immunizations. Seven days after the second and third immunizations, blood was collected from the fundus venous plexus of the mice for flow cytometric analysis. When a clear CLDN18 positive signal in the serum antibody was detected, HEK293-CLDN18.2 cells were used as the antigen for the mice. Prepare a cell suspension of 5×10 ⁷ cells/ml, and inject 100 µl into the intraperitoneal cavity of mice to boost immunity with 5×10 ⁶ cells.

Table 2 Mouse immune group Grouping Types of mice Number of mice DNA priming Cellular Boost Immunity Week 0 Week 2 Week 4 1 BALB/c, female, SPF grade, 6-8 weeks 3 Loop1, 50µg/piece 3-4 days before splenocyte fusion, intraperitoneal injection of 5×10 ⁶ HEK293-CLDN18.2 cells per mouse 2 3 Loop1-C, 50µg/piece 3 3 TML, 50µg/piece 4 SJL, female, SPF grade, 4-6 weeks 3 Loop1, 100µg/piece 5 4 Loop1-C, 100µg/piece 6 3 TML, 100µg/piece 7 C57, female, SPF grade, 6-8 weeks 3 Loop1, 25µg/piece 8 3 Loop1-C, 25µg/piece 9 3 TML, 25µg/piece

2. FACS detection of immune serum:

One week after the second and third DNA immunizations, blood from the fundus venous plexus of the mice was collected to prepare immune serum. The blocking solution (2% BSA in PBS) was diluted 1:50 in volume. Resuscitate and culture human gastric cancer cells NUGC4 (Nanjing Kebai Biotechnology Co., Ltd.) in advance, culture in 1640 medium containing 10% fetal bovine serum, 37°C, 5% CO ₂ . When the cells are in good condition and the growth confluence is 70-80%, use gastric cancer cell digestion solution (Qi's Biotechnology Co., Ltd.) to digest the cells at 37°C for 15 minutes. Complete the 1640 medium to terminate the digestion, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Add an appropriate amount of blocking solution to resuspend the cells and adjust the cell density to 1×10 ⁶ cells/ml. Mix 100µl of NUGC4 cells with 100µl of diluted mouse serum, incubate at 4°C for 1 hour; wash twice with Cell Stain Buffer (Biolegend) at 1000 rpm each time, centrifuge for 5 minutes, and discard the supernatant. Add 100 µl of PE-labeled goat anti-mouse antibody (Biolegend), incubate at 4°C for 1 hour; wash twice with Cell Stain Buffer (Biolegend), 1000 rpm each time, centrifuge for 5 minutes, and discard the supernatant. Add 300µl Cell Stain Buffer to resuspend the cells and detect with a flow analyzer.

After the second DNA immunization, mice numbered #0103 detected a significant positive signal shift, indicating that the mouse number produced a specific antibody response against CLDN18. After the third DNA immunization, positive signals were detected in all mice. The positive rate of CLDN18 in mice with numbers #0101, #0102, #0103, #0203, #0301, and #0303 was greater than 50%, indicating the third DNA immunization After immunization, these mice produced obvious antibody immune responses, and there were high levels of anti-CLDN18 antibodies in the serum.

3. Preparation of mouse hybridoma cells:

Preparation of mouse myeloma cell suspension: resuscitate mouse myeloma cells SP2/0 two weeks in advance, and culture and passage them in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO ₂ . On the day of fusion, observe the status of SP2/0 cells. The confluence of SP2/0 cells was 70%-80% by microscopic examination, and the cells were judged to be in the logarithmic growth phase. Collect SP2/0 cells by centrifugation at 300g for 5 minutes, wash once with 20ml PBS, 1000rpm, 5min, discard the supernatant; resuspend with an appropriate amount of 1640 medium, adjust the concentration of SP2/0 cells to 1×10 ⁷ cells/ml, wait use.

Preparation of mouse feeder cell suspension: the day before fusion, 20 healthy Balb/c mice aged 10-12 weeks were killed by pulling the neck. Sterile surgical scissors opened the mouse abdomen skin and cut a small cut across the abdomen. Then cut longitudinally to the chest, exposing the abdomen of the mouse. Collect peritoneal macrophages: first disinfect the mouse abdomen with alcohol cotton ball, and then use a syringe to inject 5ml of 1640 basal medium into the mouse’s abdominal cavity, gently massage the mouse’s abdomen for 5 minutes, and then suck back the abdominal cavity fluid with the syringe. Repeat once, inject 3ml of 1640 basal medium and suck back (or cut the peritoneum and suck up the remaining macrophages with a Pasteur pipette). Centrifuge the collected macrophages at 1500 rpm for 10 min, discard the supernatant, and resuspend the cells in 10 ml (1640 medium containing 10% fetal calf serum) for later use.

Preparation of mouse spleen lymphocyte suspension: 3-4 days after cell booster immunization, spleen cells of immunized mice were taken to fuse with SP2/0. The mouse was put to death by pulling the neck, the peritoneum was cut open aseptically, the spleen was taken out and the surrounding connective tissue was peeled off. Grind mouse spleen cells and filter them into a 50ml centrifuge tube with 15ml 1640 medium beforehand to prepare a single cell suspension; wash at 1500rpm, 5min, 1640 medium, discard the supernatant; add 4ml red blood cell lysate (Sigma) for lysis Red blood cells about 2min; add an equal volume of 1640 medium to 1500rpm, 5min centrifugation, discard the upper red blood cell layer; resuspend with 20ml PBS, wash twice, 1500rpm, 5min centrifugation. After the second wash, resuspend the cells with 15ml PBS. At this time, if there are clumps of connective tissue remaining, all the cells can be screened and transferred to a new 50ml centrifuge tube; for splenic lymphocyte count, use an appropriate amount of 1640 medium Resuspend and adjust splenocytes to 1×10 ⁷ cells/ml.

Take 1×10 ⁸ spleen cells and mix them with SP2/0 cells at a ratio of 1:1, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant. Resuspend and mix with BTX electrofusion buffer, and perform electrofusion operation according to the instructions of the BTX electrofusion instrument. The fused cells were allowed to stand at room temperature for 30 minutes, and 1640 selective medium containing 1×HAT, 10% fetal bovine serum was added to it, and the feeder cell suspension prepared in advance was added, and a total of 200 pieces of 96-well plates were spread. Incubate at 37°C, 5% CO _{2 for} 5-7 days. Afterwards, the fusion hybridoma cells were cultured to replicate in a medium containing 1×HT.

Example 5 Preparation of positive cell lines

a. Construction of CHO-K1-CLDN18.2 stable cell line

Resuscitate CHO-K1 cells (Shanghai Cell Institute) one week in advance, and culture them with DME/F12 complete medium containing 10% fetal calf serum at 37°C and 5% CO ₂ . The plastid of the transfected cells is pcDNA3.1-CLDN18.2-FL (see SEQ ID NO 6 for the synthetic sequence). On the day of transfection, the growth density of CHO-K1 cells reached 80%-90%. According to the operating instructions of the Biorad Gene Pulser Xcell electroporation instrument, the plastids were transferred into CHO-K1 cells, and all the transfected cells were transferred to preheated Place the culture medium in a petri dish at 37°C and 5% CO ₂ for overnight culture. On the second day, G418 (Sigma) with a final concentration of 1 mg/ml was added for resistance screening for 3-4 days, and complete medium containing G418 resistance was used for at least 5 generations to stabilize the replication, and then the expression of CLDN18.2 cells was detected. The results showed that the positive antibody IMAB362-similar-hG1 (final concentration 10µg/ml) was used to incubate the cells to detect the flow cytometry. Compared with CHO-K1 empty cells, the transfected cells showed that after 5 generations, the transfected cells stably displayed human Claudin 18.2.

b. Construction of CHO-K1-CLDN18.1 stable cell line

Using pLVX-CLDN18.1-ZsGreen plastid as template, design primer sequences SEQ ID NO 15 and SEQ ID NO 16, and prepare PCR system according to PrimeStar (Takara) instructions. Set the PCR program to denature at 98°C for 30 seconds, and anneal at 60°C for 30 seconds. , Extended for 1 minute at 72°C, and amplified by 30 cycles to obtain the CLDN18.1-FL target gene fragment (see SEQ ID NO 17). BamHI and EcoRI double enzyme digestion of the target gene and pcDNA3.1(+) vector, and ligation with T4 ligase (Takara) at 16°C overnight. The ligation product was transformed into E.coli Escherichia coli, and the plastid pcDNA3.1-CLDN18.1 was extracted with a small amount extraction kit. The plastids were electrotransfected into CHO-K1 cells according to the above steps, and the cells were cultured and subcultured in G418-resistant medium. WesternBlot results showed that the transfected cells could be stained with anti-CLDN18 antibody (Thermo) to be positive, while FACS analysis of the transfected cells After incubating with CLDN18.2 positive antibody IMAB362-similar-hG1 at a concentration of 10μg/ml, it showed a negative fluorescence signal. It showed that the transfected CLDN18.1 cells were successfully constructed.

Example 6 Screening of maternally replicated hybridoma antibodies

The first round of primary screening was performed on the parental replica hybridoma cells obtained after the fusion. The screening methods included cell ELISA and FACS to detect the hybridoma cell culture supernatant, and the CLDN18-positive hybridoma parental replicas were screened. After the initial screening showed that CLDN18 positive replications were expanded and cultured, the second round of rescreening was performed, from which FACS showed CLDN18.2 positive and CLDN18.1 negative hybridoma parent replications. After two rounds of cell ELISA and FACS screening of hybridoma antibodies, a total of 9 strains of parental replicating cells were screened from hundreds of parental replicating cells to secrete CLDN18.2 specific antibodies, which can positively bind to HEK293-CLDN18.2 cells and bind to HEK293 -CLDN18.1 cell binding is negative.

Example 7 Cultivation and screening of sub-replication

1. Cultivation of sub-replication

Single replicates were prepared from the hybridoma cells screened in Example 6. Resuspend the hybridoma cells in the 24-well plate to prepare a cell suspension. Count each replicated cell, adjust the final cell concentration to 1×10 ⁴ cells/ml in 1640 complete medium; add 60µl of cell suspension to 140µl 1640 complete medium for a total of 200µl, mix well; remove the cell suspension at -20℃ one day in advance Semi-solid medium D (StemCell), thaw at 4°C, and place it at 37°C for 1 hour before use. Take 2.5ml of semi-solid medium and 200µl of cell suspension and mix well, and let stand at room temperature for 15 minutes; transfer the mixed semi-solid medium cell suspension to a 6-well cell culture plate (Corning), inoculate 600 cells per well Cells, add appropriate amount of sterile water around the wells, and place them at 37°C and 5% CO ₂ for 5-7 days. When the cells in the well plate grow into a dense spherical single-replicating cell colony, use a 10µl pipette tip to aspirate and transfer to 1640 medium spread with 200µl 1×HT, 10% CloneEasy and 10% fetal calf serum, and mix the cell suspension thoroughly. Incubate at 37°C, 5% CO _{2 for} 2-3 days.

2. Sub-replication screening

To identify the CLDN18.2 specificity of sub-replicating cell culture supernatant antibodies, the screening method used was cell ELISA to detect the positive binding of supernatant antibodies to CLDN18.2 and CLDN18.1 stable transfected cells. The specific implementation method is as follows:

HEK293-CLDN18.1 and HEK293-CLDN18.2 cells were cultured with DMEM complete medium containing 10% fetal bovine serum at 37°C and 5% CO ₂ . When the cell growth confluence reaches 80%-90%, 0.5mM EDTA-PBS non-trypsin digestion solution digests the cells at room temperature, 1000rpm, 5 minutes centrifugation, discard the supernatant. Resuspend in DMEM complete medium and adjust the cell concentration to 6×10 ⁵ cells/ml. Spread the cells flat on a 96-well cell dish (Corning) pre-coated with polylysine, and incubate at 37°C, 5% CO _{2 for} 24 hours with 100 µl per well, so that the cells evenly cover the bottom of the well. Aspirate the culture supernatant, fix the cells with 4% paraformaldehyde at room temperature for 20 minutes, and wash 3 times with 200µl PBS for 2 minutes each time. 200µl 3%BSA-PBS to seal the well plate, 37℃, 1 hour. Add 100μl of subreplication culture supernatant to the well plate, incubate at 37°C for 1 hour, and wash 5 times with PBS. HRP-anti mouse IgG+IgM (Jackson) diluted 1:10000 in blocking solution was incubated at 37°C for 1 hour. Wash 5 times with PBS. Add 100µl TMB one-component color developing solution at room temperature and avoid light for 5-10 minutes, stop with 2M sulfuric acid, and read the OD450 of the microplate reader. The results are shown in Table 3. The hybridoma culture supernatant and CLDN18.2 combined with strong positive sub-replication (that is, the OD value is 50% higher than the positive control OD value) including: S3F10C5, S5H3A6, S5H3B6, S5H3D6, S6F10E7, S6H9B8, S6H9C8, C40B10D9, C42B12D10 and C58B11E11. Among these sub-replications, S5H3A6, S5H3B6, S5H3D6, C40B10D9, C42B12D10, and C58B11E11 showed negative binding to CLDN18.1 cells.

Table 3 Cell ELISA screening sub-replication results Serial number Subcopy number OD450 (CLDN18.2) OD450 (CLDN18.1) 1 mG2a (positive control) 1.286 0.168 2 B4B10G3 0.343 0.200 3 S3F10B5 0.615 0.198 4 S3F10C5 0.948 0.278 5 S3F10E5 0.504 0.213 6 S5H3A6 1.928 0.192 7 S5H3B6 1.379 0.152 8 S5H3D6 1.636 0.166 9 S6F10E7 0.818 0.350 10 S6F10H7 0.452 0.205 11 S6H9B8 2.353 1.495 12 S6H9C8 2.165 1.303 13 C40B10D9 0.664 0.114 14 C42B12D10 0.760 0.172 15 C58B11E11 0.671 0.197

The sub-replicas B4B10G3, S5H3B6, S6H9B8, C40B10D9, C42B12D10 and C58B11E11 were replicated and expanded. Resuspend the cells in the 96-well plate with a 200µL pipette, transfer all the cell suspension to the 24-well cell plate (Corning), add 1ml of 1640 medium containing 2% CloneEasy, 10% fetal bovine serum, and place it at 37°C. Incubate in a% CO ₂ incubator for 3-4 days.

Example 8 Identification of antibody subtypes

According to the instructions of the mouse antibody subtype identification kit SBA Clonotyping System (SouthernBiotech), add the sub-replicating cell culture supernatant to be tested to identify the subtype of the mouse antibody. The identification results are as follows:

Sub-replication B4B10G3, antibody heavy chain constant region subtype is murine IgG2b; antibody light chain constant region subtype is murine Kappa chain;

Sub-replication S5H3B6, the subtype of the constant region of the antibody heavy chain is murine IgM, and the subtype of the constant region of the antibody light chain is the murine Kappa chain;

Sub-replication S6H9B8, the subtype of the constant region of the antibody heavy chain is murine IgG3, and the subtype of the constant region of the antibody light chain is the murine Kappa chain;

Sub-replication C58B11E11, the subtype of the constant region of the antibody heavy chain is murine IgG2c, and the subtype of the constant region of the antibody light chain is the murine Kappa chain;

Sub-replication C40B10D9, the subtype of the constant region of the antibody heavy chain is murine IgM, and the subtype of the constant region of the antibody light chain is the murine Kappa chain;

Sub-replication C42B12D10, the subtype of the constant region of the antibody heavy chain is murine IgM, and the subtype of the constant region of the antibody light chain is the murine Kappa chain.

Example 9 Sequencing of antibody variable regions

Collect single-replicating hybridoma cells in a 24-well plate, adjust to 1×10 ⁶ cells/ml, 1 ml in total, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Follow the instructions of QIAGEN's RNA extraction kit (Cat:#74134) to extract a small amount of total cell RNA, and use the NanoDrop nucleic acid quantitative analyzer to determine the RNA concentration. Next, according to the Takera Reverse Transcription Kit (Cat:#6110A), 2μg of total RNA was reverse transcribed into cDNA and stored at -80°C for later use. Using cDNA as a template, the PCR system for sequencing antibody light and heavy chains was prepared and the PCR instrument program was set up according to Takara's PremixTaq enzyme operating instructions. The primers used in the PCR system were synthesized from Suzhou Jinweizhi Company. The specific primer sequences are as follows:

The heavy chain sequencing primer VH-F includes:

VH-F1 (including primer sequences SEQ ID NO 18 and SEQ ID NO 19), VH-F2 (including primer sequences SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, and primer sequences SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25), VH-F3 (primer sequence SEQ ID NO 26)

The heavy chain sequencing primer VH-R includes:

VH-R1 (primer sequence SEQ ID NO 27), VH-R2 (primer sequence SEQ ID NO 28)

The light chain sequencing primer VL-F includes:

VL-F1 (primer sequence SEQ ID NO 29), VL-F2 (including primer sequence SEQ ID NO 30 and SEQ ID NO 31), VL-F3 (including primer sequence SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34 )

Light chain sequencing primer VL-R includes:

VL-R (primer sequence SEQ ID NO 35)

The PCR products obtained by PCR amplification were entrusted to Suzhou Jinweizhi Company for sequencing, and the sequencing results are shown in Table 4.

Table 4 List of sub-replicated nucleotide sequence Table 4 List of sub-replicated nucleotide sequence Sub-replication Nucleotide sequence Heavy chain variable region Light chain variable region B4B10G3 SEQ ID NO 36 SEQ ID NO 37 S5H3B6 SEQ ID NO 38 SEQ ID NO 39 S6H9B8 SEQ ID NO 40 SEQ ID NO 41 C58B11E11 SEQ ID NO 42 SEQ ID NO 43 C40B10D9 SEQ ID NO 44 SEQ ID NO 45 C42B12D10 SEQ ID NO 46 SEQ ID NO 47

Example 10 Preparation of chimeric antibody

According to the murine antibody variable region sequence obtained in Example 9, the human IgG1 constant region and the mouse antibody variable region were combined to construct a human-mouse chimeric antibody through gene synthesis to further study antibody affinity and antibody properties.

a. Human-mouse chimeric antibody gene synthesis

Entrusted Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize chimeric antibody gene optimization and construct it into the eukaryotic expression vector pcDNA3.1(+). The chimeric antibodies are named 1CHI, 3CHI, 7CHI, 17CHI, 18CHI, 19CHI. The antibody variable region sequence corresponding to each chimeric antibody is shown in Table 5 below: Table 5 Chimeric antibody sequence Monoclonal antibody Chimeric antibody Amino acid sequence Nucleotide sequence Heavy chain variable region Light chain variable region Heavy chain variable region Light chain variable region B4B10G3 1CHI SEQ ID NO 48 SEQ ID NO 49 SEQ ID NO 36 SEQ ID NO 37 S5H3B6 3CHI SEQ ID NO 50 SEQ ID NO 51 SEQ ID NO 52 SEQ ID NO 53 S6H9B8 7CHI SEQ ID NO 54 SEQ ID NO 55 SEQ ID NO 56 SEQ ID NO 57 C58B11E11 17CHI SEQ ID NO 58 SEQ ID NO 59 SEQ ID NO 60 SEQ ID NO 61 C40B10D9 18CHI SEQ ID NO 62 SEQ ID NO 63 SEQ ID NO 64 SEQ ID NO 65 C42B12D10 19CHI SEQ ID NO 66 SEQ ID NO 67 SEQ ID NO 68 SEQ ID NO 69

b. Chimeric antibody expression and protein purification

The plastids containing the above chimeric antibody light and heavy chain genes were transiently transfected into HEK293E cells (Shanghai Institute of Cells) at a ratio of 1:1. Rotation speed 110rpm, 37℃, 8% CO ₂ shaking culture for 7 days. Centrifuge at 4500 rpm for 20 minutes to harvest the supernatant protein. Purified by ProteinA affinity chromatography: clean the AKTA chromatography system and the column with 0.1M NaOH solution for 30 min, rinse with ultrapure water; equilibrate 10 column volumes with 20mM sodium phosphate buffer pH7.4; set a flow rate of 2 ml/min , Load the sample; equilibrate the column to wash 10 column volumes; eluate 3-5 column volumes with 50mM acetic acid buffer pH3.4 to collect the eluted protein; ultrafiltration concentration replaces the antibody with 50mM acetic acid pH5.55 Store in salt buffer.

Among them, the SDS-PAGE gel electrophoresis of the chimeric antibodies 3CHI, 18CHI and 19CHI showed that the SDS-PAGE purity of the chimeric antibodies 3CHI, 18CHI and 19CHI were all greater than 90%.

Example 11 Determination of specific binding between chimeric antibody and CLDN18.2

Two weeks in advance, HEK293-CLDN18.2 and HEK293-CLDN18.1 cells were resuscitated and cultured in DMEM complete medium containing 10% fetal bovine serum at 37°C and 5% CO ₂ . When the cell growth confluence reaches 80%-90%, digest the cells with 0.5mM EDTA-PBS (Sigma) at room temperature, centrifuge at 1000rpm for 5 minutes, and discard the supernatant. Resuspend in DMEM complete medium and adjust the cell concentration to 6×10 ⁵ cells/ml. The cells were plated on a 96-well cell plate pre-coated with 2.5μg/well of polylysine (Corning), 100μl per well, 37°C, 5% CO _{2 and} incubated for 24 hours to make the cells grow evenly at the bottom of the well . Aspirate the culture supernatant, fix the cells with 4% paraformaldehyde at room temperature for 20 minutes, and wash 3 times with 200µl PBS for 2 minutes each time. 200µl 3%BSA-PBS to seal the well plate, 37℃, 1 hour. Dilute each chimeric antibody with 3%BSA-PBS to a concentration of 6μg/ml, and dilute in a 6-fold gradient to a total of 7 concentrations. At the same time, IMAB362-similar-hG1 diluted in the same concentration was used as a positive control, and human IgG1, kappa isotype control antibody (hereinafter referred to as Isotype, provided by Sino-American Crown Biotechnology (Taicang) Co., Ltd.) as a negative control, were added to HEK293 -CLDN18.2 and HEK293-CLDN18.1 cell dish. Incubate at 37°C for 1 hour, wash 5 times with PBS. HRP-anti human IgG (Jackson) diluted 1:10000 in blocking solution was incubated at 37°C for 1 hour. Wash 5 times with PBS. Add 100µl TMB one-component color developing solution at room temperature for 5-10 minutes, add 50µl 2M sulfuric acid to stop, the OD450 reading of the microplate reader. Data processing: Substitute the original data into GraphPad 5.0 software for drawing and calculation. The results are shown in Table 6.

Table 6 Determination of specific binding between chimeric antibody and CLDN18.2 Antibody number EC50 (nM) Positive control (IMAB362-similar-hG1) 0.09 Negative control (Isotype) NA 1CHI 0.55 3CHI 0.08 17CHI 2.44 18CHI 1.75 19CHI 29.90

The binding of each chimeric antibody to CLDN18.2 cells and CLDN18.1 cells is shown in Figure 1 and Figure 2, where isotype is the negative control and IMAB362-similar-hG1 is the positive control. The results showed that the binding curve of each chimeric antibody was concentration gradient dependent. Among them, 17CHI showed strong positive binding to CLDN18.2 and CLDN18.1, 19CHI showed weak positive binding to CLDN18.2 and CLDN18.1, while 1CHI, 3CHI and 18CHI antibodies could specifically bind to CLDN18.2. Does not combine with CLDN 18.1. At the same time, among the anti-CLDN18.2 specific antibodies, the EC50 value of the 3CHI antibody is small, and the EC50 value of the positive control antibody IMAB362-similar-hG1 is similar, showing strong specific binding activity with CLDN18.2.

Example 12 Affinity maturation of chimeric antibodies

In Example 11, the chimeric antibody 3CHI with the closest affinity to the positive control antibody was selected for affinity maturation and screening in vitro. The phage Fab antibody library is mainly used to screen and obtain antibodies that can maintain the specificity of CLDN18.2 while increasing the affinity.

The results of the phage screening are shown in Figure 3. The phage antibodies C1-22, A1-18, etc. all showed higher CLDN18.2 binding activity than the original 3CHI phage antibody (WT). The phages were sequenced to obtain CDR sequences with amino acid site mutations, and these mutation sites were recombined to construct a chimeric antibody with a human IgG1 constant region. After renumbering and naming the chimeric antibodies derived from the phage, the cell ELISA was used to identify whether the above 30 chimeric antibodies specifically bind to CLDN 18.2. The identification results are shown in Table 7. The table is consistent with binding to CLDN 18.1 There are 18 specific antibodies that are negative and positively bind to CLDN 18.2, and their amino acid sequences are shown in Table 11.

Table 7 Screening results of chimeric antibodies derived from phage Identification content Antibody name CLDN18.1 positive and CLDN18.2 positive 310CHI, 311CHI, 314CHI, 316CHI, 317CHI, 318CHI, 320CHI, 323CHI, 325CHI, 326CHI, 327CHI, 329CHI CLDN18.1 negative and CLDN18.2 positive 301CHI, 302CHI, 303CHI, 304CHI, 305CHI, 306CHI, 307CHI, 308CHI, 309CHI, 312CHI, 313CHI, 315CHI, 319CHI, 321CHI, 322CHI, 324CHI, 328CHI, 330CHI

Table 8 Amino acid sequence list of specific chimeric antibody Serial number Antibody name Heavy chain variable region Light chain variable region 1 301CHI SEQ ID NO 50 SEQ ID NO 70 2 302CHI SEQ ID NO 50 SEQ ID NO 71 3 303CHI SEQ ID NO 72 SEQ ID NO 73 4 304CHI SEQ ID NO 74 SEQ ID NO 75 5 305CHI SEQ ID NO 50 SEQ ID NO 76 6 306CHI SEQ ID NO 77 SEQ ID NO 78 7 307CHI SEQ ID NO 74 SEQ ID NO 70 8 308CHI SEQ ID NO 74 SEQ ID NO 71 9 309CHI SEQ ID NO 74 SEQ ID NO 76 10 312CHI SEQ ID NO 72 SEQ ID NO 76 11 313CHI SEQ ID NO 77 SEQ ID NO 70 12 315CHI SEQ ID NO 77 SEQ ID NO 76 13 319CHI SEQ ID NO 79 SEQ ID NO 70 14 321CHI SEQ ID NO 79 SEQ ID NO 76 15 322CHI SEQ ID NO 50 SEQ ID NO 80 16 324CHI SEQ ID NO 50 SEQ ID NO 81 17 328CHI SEQ ID NO 79 SEQ ID NO 80 18 330CHI SEQ ID NO 79 SEQ ID NO 81

The human gastric cancer cell NUGC4 and the stable cell CHO-K1-CLDN18.1 (prepared in Example 5) were used to verify the specificity and affinity of the above-mentioned anti-CLDN18.2 antibody on a cellular level by FACS technology. The results are shown in Table 9. After in vitro antibody affinity maturation and screening, compared with the original 3CHI antibody, each antibody can bind to the CLDN18.2 target on NUGC4 cells, but not CHO-K1-CLDN18.1 cells. . According to the degree of antibody affinity indicated by the MFI (mean fluorescence intensity) of FACS, compared with the positive control antibody IMAB362-similar-hG1, the 330CHI antibody showed a significant increase in its affinity for targeting NUGC4 cells.

Table 9 FACS test results of affinity maturation antibodies Antibody number MFI (CLDN18.2) MFI (CLDN18.1) Isotype 93 219 IMAB362-similar-hG1 2179 59 3CHI 2509 76 17CHI NA 14455 301CHI 44420 114 303CHI 38241 85 305CHI 38913 68 309CHI 42495 78 315CHI 42301 65 321CHI 46327 299 322CHI 44777 57 324CHI 36030 248 330CHI 63666 49

Verify the affinity of the above-mentioned anti-CLDN18.2 antibody in terms of protein level through the Fortebio molecular interaction instrument: first, prepare the antibody solution and the antigen dilution, and use SD buffer (PBS solution containing 0.02% Tween20 and 0.1% BSA) to chimeric The antibody is diluted to a final concentration of 5µg/ml; the antigen protein Claudin18.2-His is diluted in a 4-fold concentration gradient with SD buffer to make the concentration respectively 10µg/ml, 2.5µg/ml, 0.625µg/ml, 0µg/ml . Afterwards, select the AHC sensor to solidify the antibody, then combine with different concentrations of antigen, and perform affinity determination according to the operating instructions of Octet RED96.

The results showed that the equilibrium dissociation constant (KD) of the positive control antibody IMAB362-similar-hG1 binding to CLDN18.2 protein at 37°C was 7.14 nM, and the KD values of 330CHI and 305CHI antibodies were 0.84nM and 0.87nM, respectively, compared with the positive control Compared with that, its antibody affinity is significantly increased by an order of magnitude (see Table 10).

Table 10 Affinity test results of affinity maturation antibodies Serial number Antibody name Sensor type KD(M) Kon(M ^-1 ·s ^-1 ) Kdis(s ^-1 ) 1 IMAB362-similar-hG1 AHC 7.14×10 ^-9 5.59×10 ⁴ 3.99×10 ^-4 2 330CHI 8.40×10 ^-10 1.39×10 ⁵ 1.17×10 ^-4 3 305CHI 8.65×10 ^-10 5.13×10 ⁴ 4.44×10 ^-5

Example 13 In vitro ADCC activity determination of chimeric antibody

The day before the assay experiment, digest the CHO-K1-CLDN18.2 (prepared in Example 5) cells, resuspend the cells in complete medium, count the cells, and adjust the density to 1×10 ⁵ cells/ml, 100 μl/well Inoculate a 96-well plate and incubate overnight in a 37°C, 5% CO ₂ incubator; on the day of the test, incubate the phenol red-free 1640 medium (Gibco) to 37°C, and dilute the antibody in a 5-fold gradient; remove the plated CHO- K1-CLDN18.2 96-well plate, discard the culture medium, add 25μl of phenol red-free 1640 to each well, add the diluted antibody to the sample well, so that the final antibody concentration is 10μg/ml, 2μg/ml, 0.4μg /ml, 0.08µg/ml, 0.016µg/ml and 0.0032µg/ml; add the same amount of IMAB362-similar-hG1 and human IgG1 isotype control antibody (CrownBio) to the control wells, in a 37℃, 5% CO ₂ incubator Incubate for 1h;

Jurkat cells (hereinafter referred to as Jurkat-CD16A/NF) expressing human CD16A protein and NFAT reporter gene protein were used as effector cells, transferred to a centrifuge tube, centrifuged at 1000 rpm for 5 min at room temperature, and the supernatant was discarded; 5ml without phenol red 1640 Resuspend and wash the medium once, centrifuge at 1000 rpm for 5 min, and discard the supernatant; resuspend the cells without phenol red 1640 and adjust the cell density to 8×10 ⁶ cells/ml;

According to the effective target ratio of 20:1 (that is, the ratio of the number of effector cells to target cells is 20:1), add 2×10 ⁵ Jurkat-CD16A/NF cells, 200g, and centrifuge for 2 minutes to make the target cells effective. Full contact; incubate in a 37°C, 5% CO ₂ incubator for 5-6 hours; 30 minutes before the test, return the cell plate to room temperature, and follow the Bio-Turbo Firefly Luciferase Analysis Kit (Jiangsu Ruian Biotechnology Co., Ltd. ) Add detection reagents in the operating instructions, transfer the supernatant to be tested to a white flat-bottomed 96-well plate, and detect the fluorescence signal of the well plate with a Thermo Floskan Ascent FL fluorescence microplate reader. Substituting the original data into Graphpad 5.0 software for calculation and analysis, the results are shown in Table 11.

Table 11 In vitro ADCC activity measurement results of chimeric antibodies Group EC50 (nM) Top Isotype 13.48 6190 IMAB362-similar-hG1 0.72 215966 3CHI 0.54 211611 305CHI 0.28 215996 330CHI 0.22 214800

Compared with the Isotype negative control, the fluorescence signal of each chimeric antibody was dose-dependent with the concentration gradient. In the experimental group, the EC50 of the chimeric antibody 330CHI was 0.22nM and the EC50 of the chimeric antibody 305CHI was 0.28nM. The ADCC EC50 activity of the antibody was more than 3 times higher than that of the positive control antibody IMAB362-similar-hG1 (0.72nM). It can be seen that while the cell binding activity and affinity of the chimeric antibody against CLDN18.2 are improved, it can also effectively improve the ADCC activity of the antibody in vitro.

Example 14 Humanization of antibodies

The humanized sequence optimization of the antibody 330CHI modified by in vitro affinity maturation in Example 12 was performed. Humanized sequence optimization and antibody identification of physical and chemical properties were commissioned by Hangzhou Haoyang Biotechnology Co., Ltd. Humanized sequence optimization. Through the immune gene database (IMGT), confirm the source of the murine sequence of the variable region of the 3CHI antibody and 330CHI antibody. After homologous alignment, the source of the FR region of the heavy chain variable region sequence of the 3CHI antibody and 330CHI antibody It is derived from the mouse antibody IGHV5-15*01; the FR sequence of the light chain variable region of the antibody is derived from the mouse antibody IGKV8-19*01. The mouse antibody CDR region and the human antibody FR region are recombined to form a humanized antibody, where the human antibody light and heavy chain template is 65% or more similar to the corresponding mouse anti variable region sequence. See the source of human antibody Table 12. The name and sequence of the final humanized antibody are shown in Table 13.

Table 12 Humanized sequence design information Humanized design of heavy chain Human sequence source Similarity Is the CDR length consistent with the mouse antibody Recombinant Humanized Sequence IGHV3-48*01 77.55% Yes SEQ ID NO 82 IGHV3-23*01 75.51% Yes SEQ ID NO 88 Light chain humanized design Human sequence source Similarity Is the CDR length consistent with the mouse antibody Recombinant Humanized Sequence IGKV4-1*01 81.91% Yes SEQ ID NO 84 IGKV3-7*01 65.96% no SEQ ID NO 86 IGKV2-30*01 64.89% no SEQ ID NO 90

Table 13 Sequence list of humanized antibody Serial number Antibody name Amino acid sequence Nucleotide sequence Heavy chain variable region Light chain variable region Heavy chain variable region Light chain variable region 1 Hu330-1 SEQ ID NO 82 SEQ ID NO 84 SEQ ID NO 83 SEQ ID NO 85 2 Hu330-2 SEQ ID NO 82 SEQ ID NO 86 SEQ ID NO 83 SEQ ID NO 87 3 Hu330-3 SEQ ID NO 88 SEQ ID NO 84 SEQ ID NO 89 SEQ ID NO 85 4 Hu330-4 SEQ ID NO 88 SEQ ID NO 86 SEQ ID NO 89 SEQ ID NO 87 5 Hu330-5 SEQ ID NO 82 SEQ ID NO 90 SEQ ID NO 83 SEQ ID NO 91 6 Hu330-6 SEQ ID NO 88 SEQ ID NO 90 SEQ ID NO 89 SEQ ID NO 91

The plastid vectors containing humanized antibody light and heavy chain genes were co-transfected into HEK293E cells at a ratio of 1:1, and cultured with shaking at 110 rpm, 37°C, and 8% CO _{2 for} 7 days. Centrifuge at 4500 rpm for 20 minutes to harvest the supernatant protein. Purified by Protein A affinity chromatography: clean the AKTA chromatography system and the column with 0.1M NaOH solution for 30 min, rinse with ultrapure water; equilibrate 10 column volumes with 20mM sodium phosphate buffer pH7.4; set a flow rate of 2 ml/ min, load the sample; wash the column with 10 column volumes of equilibration solution; elute 3-5 column volumes with 50 mM acetate buffer at pH 3.4 to collect the eluted protein; concentrate by ultrafiltration to replace the antibody with 20 mM at pH 6.5 Store in phosphate buffer. The purity of the purified antibody was determined by size exclusion high performance liquid chromatography (SEC-HPLC). The monomer protein purity of the humanized antibodies Hu330-1, Hu330-2 and Hu330-4 all reached more than 95%. Among them, Hu330-1 As an example, the molecular weight of the antibody heavy chain is 48.72KD, and the molecular weight of the light chain is 24.21KD.

Example 15 FACS detection of the specificity and affinity of the humanized antibody binding to human CLDN 18.2 on a cellular level

Gastric cancer cell digestion solution was digested and treated CHO-K1-CLDN18.2 cells and CHO-K1-CLDN18.1 cells (prepared in Example 5), digested at 37°C for 15 minutes; terminated with complete medium, centrifuged at 1000 rpm for 5 minutes, and discarded Supernatant; resuspend the cells with 2% fetal bovine serum-PBS diluent, adjust the cells to 1×10 ⁶ cells/ml, add 100μl cell suspension to each well of the 96-well U-shaped chassis; use 2% fetal bovine serum-PBS Dilute the humanized antibody and control antibody to final concentrations of 10µg/ml, 2µg/ml, 0.4µg/ml, 0.08µg/ml and 0.016µg/ml respectively, mix with cells in equal volume, and incubate at 4°C for 1 hour; 1000rpm Centrifuge for 5 minutes and discard the supernatant; add 200µl Cell Stain Buffer (Biolegend) to each well and wash twice, 1000rpm each time, centrifuge for 5 minutes, discard the supernatant; add 100µl PE-anti human Fc antibody (Biolegend), incubate at 4°C , 1 hour; Wash Cell Stain Buffer (Biolegend) twice, 1000rpm each time, centrifuge for 5 minutes, discard the supernatant. Add 300µl Cell Stain Buffer to resuspend the cells, and detect by BD FACS Jazz flow cytometer.

The results are shown in Figure 4 and Figure 5. The humanized antibodies Hu330-1, Hu330-3, Hu330-5, and Hu330-6 all showed specific binding to CLDN18.2, while binding to CLDN18.1 cells was negative . Among them, the binding curve of the humanized antibody Hu330-1 almost completely overlaps with the chimeric antibody 330CHI, indicating that after the antibody is humanized, its affinity is not significantly different from that of the chimeric antibody, and basically maintains the same degree. The Kd value of the antibody relative equilibrium dissociation constant calculated according to the Graphpad fitting curve is shown in Table 14. The Kd value of the humanized antibody Hu330-1 is 1.7 nM, which is significantly lower than the positive control antibody (28.7 nM), indicating that the humanized antibody The binding activity to CLDN18.2 cells is about 15 times higher than the positive control antibody IMAB362-similar-hG1.

Table 14 Humanized antibody FACS determination of relative equilibrium dissociation constant Kd fitting results Serial number Antibody name Kd(nM) Hill slope Bmax(MFI) 1 IMAB362-similar-hG1 28.7 4.816 8224 2 330CHI 1.6 7.566 10760 3 Hu330-1 1.7 6.781 11321

Example 16 Fortebio detects the specificity and affinity of humanized antibody binding to human CLDN 18.2 in terms of protein level

Another method is to verify the affinity of the above-mentioned humanized antibody in terms of protein level, select the AHC sensor to solidify the antibody, and bind to different concentrations of antigen, and then perform the affinity determination according to the operating instructions of Octet RED96. The results are shown in Table 15. The equilibrium dissociation constant (KD) of the humanized antibody Hu330-1 binding to CLDN18.2 protein at 37°C is 5.26nM. Compared with the positive control, its antibody affinity is significantly increased by about 18 times. .

Table 15 Humanized antibody Fortebio determines the equilibrium dissociation constant KD value Serial number Antibody name KD(M) Kon(M ^-1 ·s ^-1 ) Kdis(s ^-1 ) 1 IMAB362-similar-hG1 9.44×10 ^-9 2.35×10 ⁵ 2.22×10 ^-3 2 330CHI 2.49×10 ^-10 1.30×10 ⁵ 3.23×10 ^-5 3 Hu330-1 5.26×10 ^-10 1.75×10 ⁵ 9.22×10 ^-5

Example 17 Determination of ADCC activity of humanized antibody in vitro by luciferase labeling

Take CHO-K1-CLDN18.2 cells (prepared in Example 5) as target cells, adjust the cell density to 1×10 ⁵ cells/ml, inoculate 100μl/well into 96-well plates, culture at 37°C, 5% CO ₂ Cultivate overnight in a box; dilute the humanized antibody in a 5-fold gradient so that the final antibody concentration is 10µg/ml, 2µg/ml, 0.4µg/ml, 0.08µg/ml, 0.016µg/ml and 0.0032µg/ml; Add the same amount of IMAB362-similar-hG1 and 330CHI chimeric antibodies to the control wells, and incubate them in an incubator at 37°C and 5% CO ₂ for 1 hour; the effector cells Jurkat-CD16A/NF according to the ratio of 20:1 to target Add 2×10 ⁵ effector cells to each well, 200g, centrifuge for 2 minutes to fully contact the target cells; incubate in a 37℃, 5% CO ₂ incubator for 5-6 hours; Bio-Turbo Firefly Luciferase Analysis Kit Detect the fluorescence signal of the orifice disk, and the test results are shown in Table 16.

Table 16 Determination of ADCC activity of humanized antibodies by luciferase labeling Group EC50 (nM) Top IMAB362-similar-hG1 0.69 63575 330CHI 0.18 64473 Hu330-1 0.12 68271 Hu330-2 0.11 65144

Compared with the IMAB362-similar-hG1 positive antibody, the EC50 of the humanized antibodies Hu330-1 and Hu330-2 was reduced by more than 6 times, and the ADCC activity was basically the same as that of the chimeric antibody. This shows that while the humanized antibody has high affinity, it also shows higher ADCC biological activity than the positive antibody.

Example 18 RTCA determination of ADCC killing activity of humanized antibody against tumor cells in vitro

Collect human peripheral blood to separate PBMC cells, add an equal volume of PBS containing 2% fetal bovine serum to dilute the blood sample; add 15ml Lymphoprep density gradient centrifugal fluid (StemCell) to a 50ml centrifuge tube, then add the diluted blood sample, turn off the centrifuge brake, Centrifuge at 1200g at room temperature for 10 minutes. After centrifugation, the albuginea layer is visible, which contains enriched PBMC cells. Aspirate the albuginea layer and transfer it to a new centrifuge tube. Follow the red blood cell lysis solution (Sigma) operating instructions to lyse the red blood cells at room temperature. Use 2mM EDTA and Wash 2 times with 2% fetal bovine serum in PBS, centrifuge at 400g at room temperature for 10 minutes, and discard the supernatant; resuspend the cells in 1640 complete medium containing 5% fetal bovine serum, and adjust the PBMC concentration to 3×10 ⁶ cells/ml; Secondly, human gastric cancer cell NUGC4 was used as the target cell, treated with the digestion solution of human gastric cancer tissue-derived cells, centrifuged at 1000 rpm for 5 minutes to collect the cells, and the supernatant was discarded; the cells were adjusted to 1.5×10 ⁵ cells/ml, and 100 µl of cell suspension was added to each well. After incubating the solution in an E-Plate 96-well plate at 37°C and 5% CO _{2 for} more than 24 hours, add antibody diluted with 1640 medium to make the final concentration of antibody 2μg/ml, 37°C and 5% CO ₂ for 1 hour; Add 3×10 ⁵ effector cells to each well with PBMC effector cells at a ratio of 20:1, 200g, and centrifuge for 2 minutes to fully contact the effector cells; incubate in a 37℃, 5% CO ₂ incubator to kill For 21 hours, when using the RTCA tester, the impedance value (Cell Index, CI) of the tumor cells in the 96-well plate was actually recorded, through the formula cell death%=[(control group CI-experimental group CI)/control group CI]×100% Calculate the tumor killing percentage of humanized antibody.

The results are shown in Figure 6. The tumor cell killing rate of each group increased with time within 8 to 21 hours of incubation for RTCA real-time effective target cells. At the 21st hour of in vitro killing, the humanized antibody Hu330-1 had a maximum killing rate of 45% on NUGC4 cells, which was 10% higher than the ADCC killing activity of IMAB362-similar-hG1 positive antibody on tumor cells (P>0.05).

In summary, the humanized antibody Hu330-1 can specifically target CLDN 18.2, and its binding activity and affinity are 15-18 times higher than the positive antibody IMAB362-similar-hG1 in terms of molecular and cellular degrees. The antibody-mediated ADCC killing effect on target cells in vitro shows the nature of antibody concentration-dependent. At lower concentrations, Hu330-1 can exert a certain killing effect on tumor cells.

Example 19 Optimization of Constant Region of Humanized Antibody

Combine the variable region sequence of the Hu330-1 humanized antibody with the IgG1 constant region sequence containing 5 amino acid site mutations to construct the humanized antibody Hu330-1-5M with Fc mutation (see Table 14 for the sequence); The positive antibody 5M containing the Fc mutation (see Table 17 for the sequence) was used as a control for the Fc segment.

Table 17 Sequence table of Fc mutant humanized antibody Serial number Antibody name Amino acid sequence Nucleotide sequence Heavy chain Light chain Heavy chain Light chain 1 Hu330-1 SEQ ID NO 92 SEQ ID NO 94 SEQ ID NO 93 SEQ ID NO 95 2 Hu330-1-5M SEQ ID NO 96 SEQ ID NO 98 SEQ ID NO 97 SEQ ID NO 99 3 5M SEQ ID NO 100 SEQ ID NO 11

FACS was used to identify the effect of Fc mutant humanized antibody binding to the target CLDN18.2. Digestion of NUGC4 cells with gastric cancer cell digestion solution, 37°C for 15 minutes; complete medium termination, 1000rpm, centrifugation for 5 minutes, and discard the supernatant; resuspend the cells with 2% fetal bovine serum-PBS diluent to adjust the cells to 1× 10 ⁶ cells/ml, add 100µl of cell suspension to each well of a 96-well U-shaped chassis; dilute the antibody with 2% fetal bovine serum-PBS to a final concentration of 5µg/ml, 1µg/ml, 0.2µg/ml, 0.04µg/ ml and 0.008µg/ml, mix with cells in equal volume, incubate at 4°C for 1 hour; centrifuge at 1000rpm for 5 minutes, discard the supernatant; add 200µl Cell Stain Buffer (Biolegend) to each well and wash twice at 1000rpm for 5 minutes Centrifuge and discard the supernatant; add 100µl PE-anti human Fc antibody (Biolegend), incubate at 4°C for 1 hour; wash twice with Cell Stain Buffer (Biolegend), centrifuge at 1000 rpm for 5 minutes, discard the supernatant. Add 300µl Cell Stain Buffer to resuspend the cells, and test with BD FACS Jazz flow analyzer. The results are shown in Table 18.

Table 18 FACS detection of Fc optimized humanized antibody binding activity Group EC50 (nM) Top (%) IMAB362-similar-hG1 9.78 58.8 Hu330-1 0.80 92.1 Hu330-1-5M 0.76 92.4

The positive rate of Fc mutant humanized antibody binding to NUGC4 target cells gradually saturates with the increase of antibody concentration. Its binding curve almost completely overlaps with that of humanized antibody, and it is similar to the positive control antibody IMAB362-similar-hG1 Compared with the Hu330-1-5M antibody, the binding activity of the Hu330-1-5M antibody is increased by 15 times, which indicates that the amino acid mutation of the Fc fragment of the constant region does not change the specificity and binding activity of the antibody against the CLDN18.2 target.

Example 20 In vitro ADCC killing activity detection of optimized humanized antibody

1. Measure the ADCC activity of humanized antibody with Fc mutation in vitro by luciferase labeling:

Take CHO-K1-CLDN18.2 cells (prepared in Example 5) as target cells, adjust the cell density to 1×10 ⁵ cells/ml, inoculate 100μl/well into 96-well plates, culture at 37°C, 5% CO ₂ Incubate overnight in the incubator; dilute the antibody in a 5-fold gradient to make the final concentrations of 10µg/ml, 2µg/ml, 0.4µg/ml, 0.08µg/ml, 0.016µg/ml and 0.0032µg/ml; at 37℃, Incubate with 5% CO ₂ for 1 hour; the effector cells Jurkat-CD16A/NF according to the effective target ratio of 20:1, add 2×10 ⁵ effector cells to each well, 200g, centrifuge for 2 minutes, so that the effective target cells are fully contacted Incubate in a 37°C, 5% CO ₂ incubator for 5-6 hours; Bio-Turbo firefly luciferase assay kit detects the fluorescence signal of the well plate, and the results are shown in Table 19.

Table 19 Determination of ADCC activity of Fc-optimized humanized antibodies by luciferase method Group EC50 (nM) Top IMAB362-similar-hG1 1.49 89555 Hu330-1 0.45 89817 Hu330-1-5M 0.17 100434 5M 0.44 98368

Compared with IMAB362-similar-hG1, the Fc mutation positive antibody (5M) and the humanized Hu330-1 antibody increased ADCC activity by 3 times, while the EC50 of Hu330-1-5M antibody after Fc mutation was 0.17 nM, which was higher than the positive control antibody. The ADCC activity of IMAB362-similar-hG1 is increased by more than 8 times, which obviously optimizes the in vitro ADCC enhancement effect.

2. Using LDH method to determine the ADCC killing activity of Fc mutant humanized antibodies on tumor cells in vitro:

Collect human peripheral blood to separate PBMC cells as effector cells, add an equal volume of PBS containing 2% fetal bovine serum to dilute the blood sample; add 15ml Lymphoprep density gradient centrifugal fluid (StemCell) to a 50ml centrifuge tube, then add the diluted blood sample, turn off the centrifugation Machine brake, centrifuge at 1200g at room temperature for 10 minutes. After centrifugation, the albuginea layer is visible, which contains enriched PBMC cells. Aspirate the albuginea layer and transfer it to a new centrifuge tube. Follow the red blood cell lysis solution (Sigma) operating instructions to lyse the red blood cells at room temperature. Use 2mM EDTA and Wash 2 times with 2% fetal bovine serum in PBS, centrifuge at 400g at room temperature for 10 minutes, and discard the supernatant; prepare 1640 (hereinafter referred to as heat-inactivated medium) containing 2% fetal bovine serum with heat-inactivated serum to resuspend the cells, and PBMC The concentration was adjusted to 3×10 ⁶ cells/ml; human gastric cancer cell NUGC4 was used as the target cell, treated with the digestion solution of human gastric cancer tissue-derived cells, and centrifuged at 1000 rpm for 5 minutes to collect the cells, and the supernatant was discarded; the cells were adjusted to 1.5×10 ⁵ cells /ml, add 100µl of cell suspension to each well in a 96-well cell dish, incubate at 37°C, 5% CO _{2 for} 24 hours, aspirate the medium, and add antibodies diluted with heat-inactivated medium to make the final concentration of the antibodies 10 µg/ml , 2µg/ml, 0.4µg/ml, 0.08µg/ml, 0.016µg/ml and 0.0032µg/ml, 37℃, 5% CO ₂ incubate for 1 hour; the effector cell PBMC according to the ratio of 20:1 Add 3×10 ⁵ effector cells to each well, 200g, and centrifuge for 2 minutes to fully contact the effect target cells; incubate in a 37°C, 5% CO ₂ incubator for 22 hours. At the end of the incubation, follow the instructions of the LDH kit (Dongren Chemical Technology (Shanghai) Co., Ltd.), add WST substrate reagent to the well plate, darken the color for 20-30 minutes, determine the OD450nm value, and substitute the original data into the formula cell death%=(absorption value of experimental group-spontaneous value of target cell-effector cell value)/(maximum value of target cell-spontaneous value of target cell) × 100%, absorption value of experimental group and all control groups = absorption value of this group-background value , The results are shown in Table 20.

Table 20 Determination of ADCC activity of humanized antibody optimized by Fc by LDH method Group EC50 (nM) Max (%) Isotype NA 52 IMAB362-similar-hG1 2.47 80.13 Hu330-1-5M 0.31 129.9

The humanized antibody Hu330-1-5M with Fc mutation can kill tumor cells NUGC4 in vitro, and its ADCC killing effect is dose-dependent with antibody concentration. The calculated EC50 (0.3nM) is higher than that of IMAB362-similar-hG1 positive antibody. 8 times; at the same time, the maximum killing rate of Hu330-1-5M to tumor cells is 130%, which shows a significantly enhanced in vitro ADCC killing effect compared to 80% of the positive antibody.

In summary, the Fc-mutated Hu330-1-5M humanized antibody, on the one hand, has a 15-fold increase in the affinity of the positive antibody IMAB362-similar-hG1 in the targeted binding ability to CLDN18.2; on the other hand, After Fc optimization, the ability of in vitro ADCC to kill tumor cells is further enhanced, and its EC50 activity is 8 times higher than that of positive antibodies, and the maximum killing effect is increased by more than 60%.

In summary, the CLDN18.2 antibody provided by the present invention has significant binding activity and affinity to CLDN18.2, and at the same time has high-efficiency ADCC killing activity on tumor cells in vitro, suggesting that the antibody has high-efficiency anti-tumor effects and can be prepared in preparation The application of anti-tumor drugs has broad market prospects.

Although the present invention has been described in detail above, those skilled in the art understand that various modifications and changes can be made to the present invention without departing from the spirit and scope of the present invention. The scope of patent application of the present invention is not limited to the detailed description above, but should belong to the scope of patent application.

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Figure 1 shows the binding of the chimeric antibody to CLDN18.2 cells, where the abscissa is the antibody concentration in ng/ml; the ordinate is the OD value at 450 nm. Figure 2 shows the binding of the chimeric antibody to CLDN18.1 cells, where the abscissa is the antibody concentration in ng/ml; the ordinate is the OD value at 450 nm. Figure 3 shows the binding of phage Fab to CLDN18.2 protein, where the abscissa is the phage titer; the ordinate is the OD value of 450 nm. Figure 4 shows the binding of humanized antibody to CHO-K1-CLDN18.2 cells, where the abscissa is the antibody concentration in mg/ml; the ordinate is the average fluorescence intensity MFI bound to the cells. Figure 5 shows the binding of humanized antibody to CHO-K1-CLDN18.1 cells, where the abscissa is the antibody concentration in mg/ml; the ordinate is the average fluorescence intensity MFI bound to the cells. Figure 6 shows the ADCC activity of humanized antibodies.

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Claims (16)

An anti-Claudin 18.2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein: (1) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein: (A1) The amino acid sequence of the HCDR1 is selected from SEQ ID NO 101, SEQ ID NO 102, SEQ ID NO 103 and an amino acid sequence having at least 85% sequence identity with them; (A2) The amino acid sequence of the HCDR2 is selected from SEQ ID NO 104, SEQ ID NO 105 and amino acid sequences having at least 85% sequence identity with them; (A3) The amino acid sequence of the HCDR3 is selected from SEQ ID NO 106, SEQ ID NO 107 and an amino acid sequence having at least 85% sequence identity with them; and (2) The light chain variable region includes LCDR1, LCDR2 and LCDR3, wherein: (A4) The amino acid sequence of LCDR1 is SEQ ID NO 108; (A5) The amino acid sequence of LCDR2 is selected from the group consisting of SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 111 and amino acid sequences having at least 85% sequence identity with them; (A6) The amino acid sequence of LCDR3 is selected from the group consisting of SEQ ID NO 112, SEQ ID NO 113, SEQ ID NO 114, SEQ ID NO 115, SEQ ID NO 116 and amino acid sequences having at least 85% sequence identity with them. The anti-Claudin 18.2 antibody or antigen-binding fragment thereof according to claim 1, wherein: (1) The heavy chain variable region comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO 101, HCDR2 with an amino acid sequence as SEQ ID NO 105, and HCDR3 with an amino acid sequence as SEQ ID NO 106; and (2) The light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO 108, LCDR2 with an amino acid sequence as shown in SEQ ID NO 111, and LCDR3 with an amino acid sequence as shown in SEQ ID NO 115. The anti-Claudin 18.2 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is a murine antibody, a chimeric antibody or a humanized antibody. The anti-Claudin 18.2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: (1) The amino acid sequence of the variable region of the heavy chain is selected from: (B1) The amino acid sequence as shown in SEQ ID NO 50, SEQ ID NO 72, SEQ ID NO 74, SEQ ID NO 77, SEQ ID NO 79, SEQ ID NO 82, SEQ ID NO 88; (B2) The amino acid sequence shown in (b1) is obtained by substituting, deleting or adding one or more amino acids and having the same or similar function as the amino acid sequence shown in (b1); and (B3) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (b1); and (2) The amino acid sequence of the light chain variable region is selected from: (B4) Such as SEQ ID NO 51, SEQ ID NO 70, SEQ ID NO 71, SEQ ID NO 73, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 78, SEQ ID NO 80, SEQ ID NO 81, SEQ The amino acid sequence shown in ID NO 84, SEQ ID NO 86, and SEQ ID NO 90; (B5) The amino acid sequence shown in (b4) is obtained by substituting, deleting or adding one or more amino acids, and having the same or similar function as the amino acid sequence shown in (b4); and (B6) An amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (b4). The anti-Claudin 18.2 antibody or antigen-binding fragment thereof according to claim 4, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO 79, and SEQ ID NO 79 is substituted, deleted or added with one or more amino acids The obtained amino acid sequence having the same function as SEQ ID NO 79 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 79, and the amino acid sequence of the light chain variable region is SEQ ID NO 81, SEQ ID NO 81. An amino acid sequence obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO 81 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 81. The anti-Claudin 18.2 antibody or antigen-binding fragment thereof according to claim 4, wherein: (1) The amino acid sequence of the variable region of the heavy chain is selected from: (C1) The amino acid sequence shown in SEQ ID NO 82 and SEQ ID NO 88; (C2) The amino acid sequence shown in (c1) is obtained by substituting, deleting or adding one or more amino acids, and having the same or similar function as the amino acid sequence shown in (c1); and (C3) an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (c1); and (2) The amino acid sequence of the light chain variable region is selected from: (C4) The amino acid sequences shown in SEQ ID NO 84, SEQ ID NO 86, and SEQ ID NO 90; (C5) The amino acid sequence shown in (c4) is obtained by substituting, deleting or adding one or more amino acids and having the same or similar function as the amino acid sequence shown in (c4); and (C6) An amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in (c4). The anti-Claudin 18.2 antibody or antigen-binding fragment thereof according to claim 6, wherein: The amino acid sequence of the heavy chain variable region is SEQ ID NO 82, SEQ ID NO 82 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO 82 or is the same as SEQ ID NO 82. An amino acid sequence with at least 85% sequence identity, and the amino acid sequence of the light chain variable region is SEQ ID NO 84. SEQ ID NO 84 is obtained by substitution, deletion or addition of one or more amino acids and is identical to SEQ ID NO. 84 functionally identical amino acid sequence or amino acid sequence having at least 85% sequence identity with SEQ ID NO 84; The amino acid sequence of the heavy chain variable region is SEQ ID NO 82, SEQ ID NO 82 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO 82 or is the same as SEQ ID NO 82. An amino acid sequence with at least 85% sequence identity, and the amino acid sequence of the light chain variable region is SEQ ID NO 86. SEQ ID NO 86 is obtained by substitution, deletion or addition of one or more amino acids and is identical to SEQ ID NO. 86 functionally identical amino acid sequence or amino acid sequence having at least 85% sequence identity with SEQ ID NO 86; The amino acid sequence of the heavy chain variable region is SEQ ID NO 88. SEQ ID NO 88 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO 88 or the same as SEQ ID NO 88. An amino acid sequence with at least 85% sequence identity, and the amino acid sequence of the light chain variable region is SEQ ID NO 84. SEQ ID NO 84 is obtained by substitution, deletion or addition of one or more amino acids and is identical to SEQ ID NO. 84 functionally identical amino acid sequence or amino acid sequence having at least 85% sequence identity with SEQ ID NO 84; The amino acid sequence of the heavy chain variable region is SEQ ID NO 88. SEQ ID NO 88 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO 88 or the same as SEQ ID NO 88. An amino acid sequence with at least 85% sequence identity, and the amino acid sequence of the light chain variable region is SEQ ID NO 86. SEQ ID NO 86 is obtained by substitution, deletion or addition of one or more amino acids and is identical to SEQ ID NO. 86 functionally identical amino acid sequence or amino acid sequence having at least 85% sequence identity with SEQ ID NO 86; The amino acid sequence of the heavy chain variable region is SEQ ID NO 82, SEQ ID NO 82 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO 82 or is the same as SEQ ID NO 82. An amino acid sequence with at least 85% sequence identity, and the amino acid sequence of the light chain variable region is SEQ ID NO 90. SEQ ID NO 90 is obtained by substitution, deletion or addition of one or more amino acids and is identical to SEQ ID NO 90 functionally identical amino acid sequence or amino acid sequence having at least 85% sequence identity with SEQ ID NO 90; or The amino acid sequence of the heavy chain variable region is SEQ ID NO 88. SEQ ID NO 88 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO 88 or the same as SEQ ID NO 88. An amino acid sequence with at least 85% sequence identity, and the amino acid sequence of the light chain variable region is SEQ ID NO 90. SEQ ID NO 90 is obtained by substitution, deletion or addition of one or more amino acids and is identical to SEQ ID NO 90 functionally identical amino acid sequence or amino acid sequence having at least 85% sequence identity with SEQ ID NO 90. The anti-Claudin 18.2 antibody or antigen-binding fragment thereof according to claim 7, wherein: The heavy chain amino acid sequence is SEQ ID NO 92, and SEQ ID NO 92 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO 92 or at least 85% of SEQ ID NO 92 The amino acid sequence of sequence identity, and the light chain amino acid sequence is SEQ ID NO 94, SEQ ID NO 94 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO 94, or with SEQ ID NO 94 has an amino acid sequence with at least 85% sequence identity; or The heavy chain amino acid sequence is SEQ ID NO 96. SEQ ID NO 96 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO 96 or at least 85% of SEQ ID NO 96. The amino acid sequence of sequence identity, and the light chain amino acid sequence is SEQ ID NO 98, SEQ ID NO 98 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO 98. SEQ ID NO 98 has an amino acid sequence with at least 85% sequence identity. An isolated nucleotide encoding the anti-Claudin 18.2 antibody or antigen-binding fragment thereof according to any one of claims 1-8. The nucleotide according to claim 9, wherein: (1) The nucleotide sequence encoding the heavy chain amino acid sequence is shown in SEQ ID NO 93 or SEQ ID NO 97; and (2) The nucleotide sequence encoding the amino acid sequence of the light chain is shown in SEQ ID NO 95 or SEQ ID NO 99. An expression vector comprising the nucleotide according to claim 9 or 10. A host cell transformed into the expression vector according to claim 11, wherein the host cell is selected from prokaryotic cells and eukaryotic cells, preferably mammalian cells. A method for preparing the anti-Claudin 18.2 antibody or antigen-binding fragment thereof according to any one of claims 1-8, comprising expressing the antibody in the host cell according to claim 12, and isolating the antibody from the host cell A step of. A pharmaceutical composition comprising the anti-Claudin 18.2 antibody or antigen-binding fragment thereof according to any one of claims 1-8 and a pharmaceutically acceptable carrier. An application of the anti-Claudin 18.2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 in the preparation of a medicine targeting Claudin 18.2. The application according to claim 15, wherein the drug targeting Claudin 18.2 is used to treat gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer.

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